In recent years, many procedures and devices have been disclosed for assay of a substance in a fluid. In particular, assay of biological fluids for substances therein has been the subject of much attention. Among the classes of substances for which assays have been disclosed are endogenous substances such as hormones and antibodies, and exogenous substances such as antigens or drugs. Such substances to be assayed are frequently present in the biological fluid at very low levels, often in the range of 10.sup.-10 molar or lower.
A particularly useful approach to assay for substances present in such low concentrations is immunoassay. In an immunoassay, advantage is taken of the specific reaction of an antigen for an antibody. Two types of immunoassay are in general use. In the first type of assay, commonly referred to as a homogeneous assay, all assay reagents are eventually combined with no separation steps necessary during the procedure. In the second type of assay, commonly referred to as a heterogeneous assay, one or more separation steps are required.
Heterogeneous assays are often performed by fixing or immobilizing one of the assay reagents to an appropriate insoluble solid support. Particularly useful solid supports are microporous membranes as described in U.S. Pat. No. 4,066,512. Such membranes may be subjected to any number of sequentially performed assay steps involving separation and washing of assay components. U.S. Pat. No. 4,200,690 to Root et al. describes assay for an antigen using an antibody immobilized on such a membrane. Assays performed in this fashion are generally referred to as flow-through assays. Separations in such assays conventionally have been performed by allowing a fluid phase to filter through the membrane by gravity. However, since such assays frequently require repeated washing between steps, an excessively long assay time may be required. Exemplary of an assay using gravity flow of liquid is U.S. Pat. No. 4,111,754 to Park.
Various methods have been developed to induce more rapid filtration. U.S. Pat. No. 4,277,560 to Gray et al. discloses a method and apparatus for performing flow-through assay by application of pressure. Chen et al., in U.S. Pat. No. 4,090,850 teaches an apparatus for radioimmunoassay in which antibody coated cellulose discs serve as a solid support in a flow-through assay using multiple wells in a receptacle plate connected to an external vacuum source. Bagshawe, in U.S. Pat. No. 3,888,629, teaches a device in which an assay membrane is in contact with an absorbant material so that filtration of assay fluids into a defined volume is facilitated by capillary action. U.S. Pat. No. 4,246,339 to Cole et al. uses capillary action as a filtration aid in a test device having membranes in multiple assay wells which can be biased into and out of contact with an absorbent material.
European Patent Application No. 0141547 discloses a device in which immunoassay reactions are carried out in a liquid phase above a filter membrane and assay reaction products are filtered using a preevacuated tube as vacuum source.
The use of vacuum to promote flow of an assay fluid through a membrane and to collect the fluid in an absorbent material localized in a defined volume was also known to Applicants at the time of the present invention.
Thus, it is known to use permeable membranes as solid supports in immunoassay procedures and a filters to remove immunoassay reaction products from fluid assay phases. It is further known that a wet filter exhibits properties related to surface tension which are similar to the properties of an array of liquid-filled capillaries and that air will not pass through a wet filter until the pressure on the filter exceeds the capillary attraction of the fluid in the pores of the filter. This pressure is conventionally referred to as the "bubble point," and is defined as the pressure required to blow the first bubble detectable by its rise through a layer of liquid covering the filter. The bubble point, which is easily determined, is a reliable measure of membrane integrity and is generally expressed in kg/cm.sup.2, lbs/in.sup.2 or bars, wherein one bar equals 14.5 lbs/in.sup.2. A general discussion of the bubble point, determination thereof and values for representative filter membranes are given in Millipore Laboratory Products Catalogue, Millipore Corporation, Bedford, Mass., 1982, pages 28 and 36-37.